The overall objective of the proposed investigation is to synthesize a variety of members of a new class of positively-charged lipids, all of which are based on the backbone structure of naturally occurring phospholipids many of which promise to be unusually effective agents for gene transfection and to elucidate the mechanism by which they interact with DNA and facilitate its entry into cells. Presently, cationic lipids are commonly used for transfection of eukaryotic cells in culture and are becoming indispensable tools in molecular biology research. Recently, medical uses of the technique have been indicated by application of the technique to whole animals in which gene transfection has been remarkably successful in introducing functional genes into a large proportion of cells in many tissues. These successes with animal models indicate that cationic lipids could become one of the primary delivery systems for gene therapy and gene therapeutics of, among others, infectious diseases and cancer. Although cationic lipids hold considerable promise as gene transfer agents, the cationic lipids most popular to date are not amenable to molecular tailoring for optimizing their efficiency. Hence, little is known about how the lipid interacts with DNA, how the resulting complex enters the cell and how the DNA is released for eventual transfer to and expression in the nucleus. We propose a study of the structure and transfection efficiency of DNA complexes formed with a new class of cationic lipids derived from natural phospholipids which can be altered synthetically to maximize transfection efficiency and which, as variants of naturally-occurring molecules, are biodegradable to preclude toxic accumulation in the organism. The first aim calls for the systematic variation of the structure of these lipids, which is facilitated by the variety of precursors available and the ease of modification of these precursors. Determination of the efficiency of transfection of tissue culture cells will reveal which aspects of molecular structure are critical for transfection and whether there are specific interactions with particular types of cells. The second aim is to elucidate the structure of the complex. Choosing compounds that vary widely in transfection efficiency, we will examine the structure of their complexes with DNA to determine what characteristics of the complex are critical for transfection. In particular, the stability of the complex will be tested to determine if it is unstable under cellular conditions, a property which would partially explain why these complexes are effective transducing agents. The third aim is to understand how the complex enters the cell, by fusion at the cell surface or by endocytosis, as well as to identify the region in the cell where the complex dissociates. Finally, the compounds will be assessed with respect to cellular toxicity and metabolic fate.